Mononuclear phagocytes in therapeutic drug delivery

ABSTRACT

The invention relates to the exploitation of the migratory behavior of mononuclear phagocytes with a view to targeting therapeutic drug delivery. The invention therefore concerns the attachment or incorporation of a therapeutic agent to or into a mononuclear phagocyte and the subsequent migration of the munonuclear phagocyte to a target area.

The invention relates to a method of drug delivery; means thereforincluding components thereof which have particular, but not exclusive,application in cancer therapy development.

Macrophages often comprise 20-60% of the tumour cell mass in breastcarcinomas and form intimate contacts with malignant cells. This haslong been thought to represent part of the host's defence mechanismsagainst the tumour; however, their function at such sites in the bodyremains an enigma at present as macrophages isolated from human ormurine tumours exhibit reduced tumouricidal and antigen-presentingactivities compared to those from normal tissues (1).

Monocytes are produced in the bloodstream and extravasate (i.e. exit)into surrounding tissues including such diseased tissues as malignanttumours and atherosclerotic plaques, where they differentiate intomacrophages and perform immune, secretory, phagocytic and otherfunctions. Monocytes and macrophages are collectively termed mononuclearphagocytes. As tissue macrophages have a lifespan of 60 to 90 days andthe number of macrophages in tumours remains constant, it is believedthat there is a constant attachment of monocytes to the tumourendothelium and influx of monocytes into the tumour cell mass.

Hypoxia, that is, very low levels of oxygen, exist only in some forms ofdiseased tissue (e.g. malignant tumours, ischaemic heart tissue etc).Hypoxia and/or hypoglycaemia is thought to occur in growing tumours whenthe increasing metabolic demands of the rapidly expanding tumour cellpopulation outstrip the supply of oxygen/glucose etc., made available tothem by simple diffusion across the tumour mass from vessels insurrounding normal tissues.

Ischaemia, that is, a deficiency of blood flow to part of the body alsoexists in some forms of diseased tissue (e.g. malignant tumours,ischaemic heart tissue etc). Malignant tumours tend to outgrow theirblood supply and often have ischaemic areas of tumour cell apoptosis andnecrosis. Moreover, the vascular supply in some tumour areas cancollapse resulting in the formation of further ischaemic areas.Ischaemic tissues are also found in coronary artery disease, diabeticretinopathy and following strokes. Cells in such areas of vascularocclusion or collapse experience various forms of stress such as glucosedeprivation, low pH, elevated levels of lactate and pyruvate, ion or cnatic imbalance and hypoxia.

Our recent and surprising data indicate that once monocytes enter atumour from the bloodstream and differentiate into macrophages, theypreferentially congregate in hypoxic (i.e. poorly vascularised andnecrotic) sites deep within a tumour mass remote from blood vessels (2).Refer to FIG. 1, which represents a bar chart of the Distribution ofMacrophages in Relation to Blood Vessels. Moreover, breast tumours, withmore hypoxic/necrotic areas, are more heavily infiltrated withmacrophages, which preferentially locate to, or around, the necroticsites (refer to FIG. 2, which represents a bar chart of the Associationof Macrophage Index with Necrosis in Breast Carcinomas). Very recently,macrophages have also been shown to congregate in ischaemic/necroticsites in ovarian tumours (3). Over a decade ago experimental hypoxia wasshown to induce the production of angiogenic factors by macrophages invitro (2). Taken together these data could underpin our recent finding,that increased numbers of macrophages in breast tumours equate withhigher levels of angiogenesis and increased fatalities in breast cancer(2).

We examined tumour biopsies from 100 breast cancer patients, and foundthat macrophage infiltration was strongly associated with reducedrelapse-free interval and overall survival—even in lymph node negativepatients alone (i.e. the better prognosis group). Indeed macrophageinfiltration proved to be almost as important in predicting outcome aslymph node status in breast cancer. We went on to see if this was due toan effect of macrophages on such important parameters of tumouraggressiveness as the mitotic index, receptor status, degree ofangiogenesis, etc. It was at this point that a highly significant andunexpected correlation between the degree of macrophage infiltrationwithin a tumour mass and angiogenesis emerged, suggesting a role forthis cell type in some or all of the steps of tumour angiogenesis (2).

Our unique observations suggest that, since the entry or presence ofmacrophages into or in such diseased tissues appears to be deleteriousto the patient, therapies specifically focused on either blocking theirentry; their destruction; and/or their exploitation to carry therapeuticagents into such diseased tissues could prove to have therapeuticbenefits.

Monoclonal antibodies have been considered for many years to be the bestway of delivering cytotoxic agents to tumours, but this approach has sofar been disappointing in clinical trials (4, 5). The main reason forthe lack of therapeutic efficacy of antibody conjugates in solid tumoursis the resistance of the turnour to penetration by macromolecules. Instudies with radiolabelled antibodies, typically only 0.001-0.01% of theinjected dose localises to each gram of solid tumours in humans (6, 7).The poor penetration of antibodies is thought to be due to a number offactors. Firstly, the antibodies must cross the physical barrier of theendothelial cell layer in tumour blood vessels and the, often dense,fibrous stroma packed between tumour cell areas. Secondly, the densepacking of tumour cells and tight junctions between epithelial tumourcells hinder the transport of the antibody within the tumour mass.Thirdly, the absence of lymphatics within the tumour contributes to thebuild up of a high interstitial pressure which opposes the influx ofmolecules into the tumour core.

One solution to the poor penetration of antibody conjugates into solidtumours would be to attack the endothelial cells in the tumour, insteadof the tumour cell themselves, which are readily accessible tointravenously injected antibody. This then leads to destruction of thetumour blood vessels and the death of neighbouring tumour cells whichrely on the blood supply for oxygen and nutrients. Early studies usingmurine models have met with some success in this area (8), but thereremains the problem in humans of how to target tumour endothelial cellsand not those in normal tissues. Furthermore, as mentioned earlier, ourwork strongly suggests the tumour necrosis resulting from this approachwill trigger compensatory angiogenic activities in tumour-infiltratingmacrophages in the vicinity. This would oppose the effects of thetherapeutic agent.

Our inventive solution is to attach the agent (e.g. tumour orendothelial cell cytotoxin) to monocytes in the peripheral blood, thatis the cells which gain entry to the tumour in large numbers to formtumour-infiltrating macrophages. Since monocytes swarm to the tumoursite in large numbers as an early and ongoing event in tumourdevelopment, they can be used to carry therapeutic agents (e.g.cytotoxic drugs or toxins) into the centre of solid tumours. In supportof this suggestion is the fact that many studies have shown thatmalignant tumours actively recruit this cell type and that monocytesthen follow a chemotactic trail produced by tumour cells. Further, wehave uniquely shown that mononuclear phagocytes congregate in tumourareas where they are most needed, i.e. hypoxic or stress areas, possiblyto help initiate angiogenesis.

Although we have described the invention with particular reference totumour cells, it can be used in any instance where mononuclearphagocytes infiltrate or are attracted to hypoxic/ischaemic/stresstissue or conditions. Thus, the invention can be used during developmentto control the vascularisation of developing tissue, typically, but notexclusively, with a view to targeting a hypoxia regulatable agent so asto promote or enhance vascularisation. Alternatively the invention canbe used to target hypoxia regulatable agents to damaged tissue, forexample to tissue where de-vascularisation has occurred following damageto the vascular system via an amputation, stroke, cardiac arrest,extreme hypertension, ischaemia, burns etc.

It follows from the information provided herein that the invention maybe used to prevent or reduce tissue vascularisation, or to promote orenhance vascularisation, or to simply deliver selected drugs tohypoxic/ischaemic/stress sites where mononuclear phagocytes aretypically present.

In addition, given our finding that mononuclear phagocytes infiltrate orare attracted to hypoxic/ischaemic/stress tissue or conditions theinvention can also be used with a view to targeting a stress regulatableagent such as an ischaemically regulatable agent. Thus, in the instancewhere mononuclear phagocytes infiltrate a site which is also ischaemicand thus characterised by glucose deprivation, low pH, elevated levelsof lactate or pyruvate, and/or hypoxia it is possible to arrange fortargeting of an ischaemically regulatable agent.

It is therefore an object of the invention to provide a novel drugdelivery system which exploits the fact that mononuclear phagocytescollect or are attracted to hypoxic or ischaemic or stress sites.

It is yet a further object of the invention to provide a regulatabledrug delivery system which enables the activation of said drug to becontrolled, and more specifically, to be controlled so as to only beactive under hypoxic and/or stress and/or ischaemic conditions.

It is yet a further object of the invention to provide a novel drug foruse in the drug delivery system of the invention.

The invention, in its broadest aspect, comprises the use of mononuclearphagocytes to deliver therapeutic agents to tissues and especiallyhypoxic and/or stress and/or ischaemic sites.

According to a first aspect of the invention there is therefore provideda therapeutic composition comprising a regulatable agent and an agentthat binds to a cell surface element of a mononuclear phagocyte.

In a preferred embodiment of the invention said binding agentpreferentially binds to said cell surface element.

Reference herein to the term preferentially is intended to include theselective or targeted binding of said agent to mononuclear phagocytes.

In a preferred embodiment said agent is a hypoxia and/or ischaemiaand/or stress regulatable agent.

It will therefore be apparent that the hypoxia and/or ischaemia and/orstress regulatable agent will be affected by hypoxic and/or ischaemicand/or stress conditions and typically affected so as to only be activein such conditions. Moreover, said binding agent, which is typicallycoupled to said regulatable agent, attaches the composition tomononuclear phagocytes and so targets the regulatable agent, to sitestypically infiltrated by mononuclear phagocytes. Thus in the instancewhere said mononuclear phagocytes penetrate hypoxic and/or ischaemicand/or stress sites said composition is suitably delivered to such sitesand the regulatable agent becomes active.

The invention is elegant in so far as the body's own mechanisms areexploited for the specific delivery of drugs but the invention is safein so far as the drugs remain inactive until exposed to hypoxic and/orischaemic and/or stress conditions.

Given the above nature of the invention agents suitable for use inmanufacturing the said composition will be known to those skilled in theart and therefore the following preferred embodiments are not intendedto be exhaustive but rather illustrative.

For example, in one embodiment of the invention said hypoxia regulatableagent may comprise a therapeutic gene, that is to say a nucleotidesequence encoding a therapeutic agent which is under the control of ahypoxia sensitive agent such as a hypoxia regulated expression elementi.e. a promoter or enhancer which is sensitive to hypoxia. Thus, underconditions of hypoxia said element will be activated so as to enable thegene encoding the therapeutic agent to be expressed. In this embodimentof the invention the binding agent comprises an agent that ensuresinternalisation of said therapeutic gene, for example, withoutlimitation a viral or a non-viral vector, with a view to incorporatingsame into the mononuclear phagocyte genome.

In addition, or alternatively, the hypoxia regulatable therapeutic genemay encode a prodrug activation enzyme, that is to say an enzyme whichconverts a relatively inactive drug into a more active one. An exampleof this kind of enzyme is thymidine phosphorylase which activates the5-FU prodrugs capcetabine and furtulon. Another example of a prodrugactivation enzyme is the herpes simplex thymidine kinase or cytosinedeaminase which, once internalised into the mononuclear phagocytes wouldact as a reservoir for activation of the prodrugs ganciclovir and5-fluorocytosine.

Another example of a prodrug activation enzyme is the P450 family e.g.CyP2B6 which activate cyclofosfamide and ifosfamide (30)

Other examples of hypoxia regulatable therapeutic genes are to be foundin PCT/GB95/00322 (WO9521927).

Alternatively said hypoxia regulatable agent may comprise abioreductively activated drug prodrug such as RSU1069 or Tirapazaminewhich are activated at very low levels of oxygen as well as with contactwith enzymes such as reductases. Thus, where not only hypoxia, butprotein action, typically enzymic, is required for activation of saidregulatable agent, said therapeutic composition of the invention mayfurther comprise an agent that activates said hypoxia regulatable agent,such as a reductase and/or a gene encoding said reductase.

In a further embodiment of the invention said ischaemia or stressregulatable agent may comprise a therapeutic gene, that is a geneencoding a therapeutic agent which is under the control of an ischaemiaor stress sensitive agent such as an ischaemia or stress regulatedexpression element i.e. a promoter or enhancer which is sensitive to anyone or more of the factors characterising ischaemia or stress such as,for example, without limitation, glucose deprivation, low pH, elevatedlevels of lactate or pyruvate and/or ion or osmotic imbalance. Thus,under conditions of ischaemia or stress said elements will be activatedso as to enable the gene encoding the therapeutic agent to be expressed.In this embodiment of the invention the binding agent comprises an agentthat ensures internalisation of said therapeutic gene with a view toincorporating same into the mononuclear phagocyte genome.

As mentioned above, the ischaemia or stress regulatable agent maycomprise a pro-drug activation enzyme as previously described, or a geneencoding same.

Suitable examples of DNA sequences known to be activated by any of theaforementioned factors, or “stress factors” may be used in the inventionand examples of such sequences include, without limitation, promoterand/or enhancer sequences activated by glucose deprivation, low pH orelevated levels of lactate or pyruvate and/or ion or osmotic imbalance.For example, the glucose-regulated proteins (grp's) such as grp78 andgrp94 are highly conserved proteins known to be induced by glucosedeprivation (19). The grp78 gene is expressed at low levels in mostnormal healthy tissues under the influence of basal level promoterelements, but has at least two critical “stress-inducible regulatoryelements” upstream of the TATA element (19, 20). Attachment to atruncated, 632-base pair sequence of the 5′ end of the grp78 promoter(which include these two stress-inducible regulatory sacs) confers highinducibility to glucose deprivation on reporter genes in vitro (20).Furthermore, use of this promoter sequence in a retroviral vector drovehigh level expression of a reporter gene in tumour cells in murinefibrosarcomas, particularly in central, relatively ischaemic/necroticsites (20). In addition it is thought that grp78 may be inducible byother stress factors (27).

Another group of highly conserved, what may be termed, “stress proteins”are those responsive to low pH. The genes for these proteins are knownto have acid-inducible promoters, examples of which have been cloned andcharacterised in recent studies (21) indeed, it was demonstrated thatthe acid-inducibility of several such defined DNA sequences in hybridDNA constructs consisting of each one of these fused to a reporter geneencoding a green fluorescent protein was sensitive to pH. Maximuminduction was seen when the pH was dropped to 4.5.

Thus, said stress regulatable agent comprises a therapeutic gene underthe control of a stress regulated expression element known to thoseskilled in the art. Notably, a stress regulated expression element isintended to include both homologous and heterologous elements i.e. thatis to say promoters and/or enhancers for genes known to be expressed, orover-expressed, by mononuclear phagocytes in stress conditions; andpromoters or enhancers for genes known to be over-expressed by othertissues in said conditions, respectively. Examples of the former groupinclude the expression element of the osteopontin gene and thymidinephosphorylase gene. Other examples of homologous promoters or enhancerscomprise those involved in the phagocytic activity of macrophagesincluding CD36, CD68, thrombospondin, the αvβ3 integin and low densitylipoprotein receptors (25, 26).

In a yet alternative embodiment of the invention said agent may beregulated by means other than hypoxia or ischaemia or stress.

Indeed, the use of hypoxia or ischaemia or stress to regulate theexpression/activity of a therapeutic gene may not be an exclusivefeature of the utilisation of mononuclear phagocytes as a means oftargeting therapeutic genes to tumours or regions ofhypoxia/ischaemia/stress.

In a further embodiment of the invention it may be preferable to placethe therapeutic gene under the control of an inducible or repressiblepromoter element, transcription from which can be modulated to regulatethe effective dose of the therapeutic gene. This may result in moreaccurate control of expression of the therapeutic gene and therebyreduce undesirable side effects as a consequence of the gene therapy.

By way of example, a constitutively active promoter used to express atherapeutic gene/agent is placed under the regulation of the antibiotictetracycline by inclusion in the promoter of the tetracycline repressorDNA sequence. The vector DNA incorporating the therapeutic promotercassette includes the DNA sequence of the tetracycline repressor proteinunder the control of a suitable promoter element. Transfection of saidDNA construct into the mononuclear phagocyte genome results inexpression of tetracycline repressor protein and a repression in thetranscription of the therapeutic gene. Transfected mononuclearphagocytes are then re-introduced into a patient to allow said phagocyteto infiltrate, for example, a tumour. Tetracycline is then administeredorally, via an intravenous route, or injected directly into the tumourtissue. The administration of tetracylcine to the patient results ininactivation of the binding between the tetracycline repressor proteinand the operator DNA sequence in the promoter of the therapeutic genethus enabling its expression. The effective dose of tetracycline can bevaried to regulate the expression of the therapeutic gene/agent andthereby optimise the effect of the therapeutic gene/agent.

The system may also be exploited to enable the regulated expression of a“suicide” gene, the expression of which in a mononuclear phagocyte wouldresult in the death of the phagocyte carrying the gene. Selectiveremoval of mononuclear phagocytes may then inhibit revascularisation oftumour tissue thereby restricting tumour development.

This example of regulated expression has been shown to occur in bothprokaryotic (WO 9532295-A) and eukaryotic cells (28, 29) to successfullyregulate the expression of both homologous and heterologous DNAsequences. This description is merely to serve as an example of how aprokaryotic sequence can be used to regulate the expression of aeukaryotic gene and is not meant to be exclusively limited to thetetracycline repressor operon.

Alternatively, or in addition, especially where internalisation of saidtherapeutic composition is required said therapeutic composition mayfurther comprise an internalisation agent so as to ensure that thetherapeutic composition is internalised by the mononuclear phagocytes.Agents which are suitable for ensuring internalisation of thetherapeutic composition include, but are not limited to, plasminogenactivation inhibitors (PAI-1 or PAI-2) or protease nexin (PN).

In yet a further preferred embodiment of the invention said bindingagent is adapted to target or bind to any one or more cell surfacemononuclear phagocyte molecules such as antigens or receptors. Further,said binding agent may comprise an antibody to any one or more of saidmolecules such as antigens or receptors, or an effective fragment ofsaid antibody. Alternatively still said binding agent may comprise asuitable ligand either synthetically manufactured or naturallyoccurring.

Alternatively, the binding agent may comprise a vector. The vector maybe a non-viral vector. Examples of non-viral vectors include plasmid DNAcompacted with a DNA compaction agent such as one containingpoly-L-lysine or a liposome or immunoliposome containing plasmid DNAcompacted with a DNA-compaction agent (such as a poly-lysine). Thenon-viral vector may be a targeted non-viral vector such as plasmid DNAcompacted with mannosylated PolyLysine (MPL).

In a yet further preferred embodiment of the invention said bindingagent may comprise a viral vector.

The vector may be a recombinant viral vector such as an adenovirusvector, an adeno—associated viral (AAV) vector, a herpes-virus vector ora retroviral vector in which case gene delivery (transduction) ismediated by viral infection. The viral vector may be a defective viralvector which is not capable of replication in the target cells. Theviral vector may also be a chimeric viral vector containing componentsof more than one virus such as retrovirus pseudotyped with the envelopeof another virus or an adenovirus capable of expressing retroviralgenetic elements (eg. Feng et al 1997 Nature Biotech. 15:866-870).Preferably the viral vector is one which is capable of preferentiallytransducing non-dividing cells such as a lentiviral vector, includingHIV based vectors (Naldini et al 1996 Science 272: 263-267).Particularly preferred is a targeted viral vector such as a targetedadenoviral vector capable of preferentially transducing mononuclearphagocytes. Examples of methods for targeting adenoviral vectors tospecific cell types are described (eg Krasnykh et al 1996 J. Virol 70:6839-6846; Wickham et al 1996 J. Virol 70: 6831-6838); Stevenson et al1997 J. Virol. 71: 4782-4790; Wickham et al. 1995 Gene Therapy 2:750-756. Also preferred is a viral vector which is resistant to humancomplement, for example by production in a human cell line.

In any event, the viral or non-viral vector will contain a promoter todirect expression of the or each therapeutic gene and may containadditional genetic elements for the efficient or regulated expression ofintroduced genes, including enhancers, translation initiation signals,internal ribosome entry sites (IRES), splicing and polyadenylationsignals. The enhancer may contain elements for regulated expression suchas a hypoxia regulated enhancer (for example a binding element for thetranscription factor HIF1) or elements which respond to stress or lowglucose. The enhancer elements or elements conferring regulatedexpression may be present in multiple copies. Combinations of suchelements are also envisaged. Genes encoding appropriate transcriptionfactors may also be included in the vector in order to enhance theresponse to hypoxia, stress or low glucose. For example a gene encodingHIF1-alpha may be included in the vector (Jiang et al 1996 J Biol Chem271: 17771-17778).

Additional vector components will be provided for other aspects ofvector function such as vector maintenance, nuclear localisation,replication, and integration as appropriate using components which arewell known in the art.

Suitable therapeutic genes may encode proteins which are secreted fromthe mononuclear phagocyte or which are retained within the cell. Fortreatment of cancer, suitable therapeutic genes include genes encodingproteins which act as for example: activators of anti-tumour immunemechanisms (such as cytokines, co-stimulatory molecules andimmunoglobulins); of angiogenesis; or which provide enhanced drugsensitivity (such as pro-drug activation enzymes). The use ofcombinations of such genes is also envisaged. Genes or combinations ofgenes which show a by-stander effect in the destruction of neighbouringor distant tumour cells are preferred. For treatment of ischaemia, genesencoding proteins which act to stimulate angiogenesis (such as vascularendothelial growth factor (VEGF)) or other growth factors are suitableexamples.

A brief list of those cell surface molecules that may be targeted bysaid binding agent is as follows; the receptor for human UrokinasePlasminogen Activator (uPAR; CD87); the receptor for human ColonyStimulating Factor (CSF-1); CD63; CD64; CD11b; CR3; CD33; the scavengerreceptor; all or part of the receptor for the various forms of humanmonocyte chemoattractant protein (MCP-1, 2, etc); CD14; mannose ormannose-6-phosphate surface receptors; CD16; or HLA-DR.

In a yet further preferred embodiment of the invention said therapeuticcomposition comprises any one or more of the drug conjugates illustratedin Table 1, and more preferably, any combination of part I and/or partII and/or part III components specified in Table 1.

According to a yet further aspect of the invention there is provided adelivery system for targeting therapeutic compositions to hypoxic and/orischaemic and/or stress sites comprising a hypoxia and/or ischaemiaand/or stress regulatable agent and an agent for controlling thefunctional effectiveness thereof, and coupled thereto, a binding agentfor a cell surface molecule of a mononuclear phagocyte.

According to a yet further aspect of the invention there is provided amethod for targeting desired agents to hypoxic and/or ischaemic and/orstress sites comprising;

-   -   (i) coupling at least one of said agents to a binding agent that        is adapted for binding or targeting a cell surface molecule        expressed by a mononuclear phagocyte;    -   (ii) exposing said coupled agent to mononuclear phagocytes; and    -   (iii) allowing said mononuclear phagocytes to migrate, under        conditions that support migration, either in vitro or in vivo.

According to a yet further aspect of the invention there is provided amethod for treating conditions associated with hypoxic and/or ischaemicand/or stress states comprising administering to an individual to betreated the therapeutic composition of the invention.

According to a yet further aspect of the invention there is provided amethod for treating conditions associated with hypoxic and/or ischaemicand/or stress states comprising withdrawing blood and/or serum from anindividual to be treated and treating said blood and/or serum in vitrowith a hypoxically and/or ischaemically and/or stress inducibletherapeutic gene under conditions that enable incorporation of said geneinto the nucleus of mononuclear phagocytes and re-injecting said treatedblood and/or serum into the individual either systemically or directlyinto a hypoxic and/or ischaemic and/or stress area. According to a yetfurther aspect of the invention there is provided mononuclear phagocyteswhich have coupled thereto, or internalised therein, at least a hypoxiaand/or ischaemia and/or stress regulatable agent and, optionally, anagent that is adapted to bind to a mononuclear phagocyte ligand which istypically found on the cell surface of said mononuclear phagocyte.

According to a yet further aspect of the invention there is provided amethod for selectively destroying a mononuclear phagocyte comprisingattaching thereto or internalizing therein a cytotoxic, hypoxicallyand/or ischaemically and/or stress activated agent and exposing saidmononuclear phagocyte to hypoxic and/or ischaemic and/or stressconditions that occur either artificially by induction or occur/existnaturally.

In the instance where hypoxia and/or ischaemic and/or stressoccurs/exists naturally said mononuclear phagocyte migrates in a normalmanner to said hypoxic and/or ischaemic and/or stress area so that thesaid agent is only activated at a target area. In this way thepotentially deleterious effects of mononuclear phagocytes in tumours isobviated. Moreover, having regard to the nature of said agent abystander effect may be achieved, for example where said cytotoxic agentis released on death of said mononuclear phagocyte it may have a furtherdeleterious effect on the hypoxic and/or ischaemic and/or stress tissue,such as, but not limited to, tumour tissue.

Many of the preferred embodiments hereinbefore described representappropriate modifications of any one or more of the above referred tofurther aspects of the invention.

Embodiments of the invention will now be described by way of exampleonly with reference to the following Figures and Table wherein:

FIG. 1 shows the distribution of macrophages in areas of high and lowvascularity in 46 invasive breast carcinomas (i.e. assessed byquantification of CD31 postivity of blood vessels; see reference 3).

FIG. 2 shows association of macrophage index with necrosis in 92 breastcarcinomas. * w.r.t Grade O group (Mann Whitney U Test). Number of datapoints in each group is indicated at the base of each column. [Gradesfor necrosis: 0, no necrosis; 1, few focal areas of necrosis; 2, manyfocal areas of necrosis; 3, almost entirely necrotic].

FIG. 3 shows the effect of clamp-induced hypoxia (induced by tumourvessel clamping for 2 h) on macrophage infiltration in human breastcancer xenografts grown in nude mice;

FIG. 4 shows the diagram of an Adenovirus transfer vector (QBI-HRE-LacZ)containing a LacZ gene under the control of a hypoxia regulatedpromoter-enhancer;

FIG. 5A-5C. Macrophage infiltration into hypoxic areas in tumourspheroids (i.e. an in vitro model of tumour hypoxia).

-   -   FIG. 5A shows the oxygen profile across a tumour cell spheroid.        All but the cells in the outer 100 μm of these 3-D cultures are        hypoxic (i.e. experiencing oxygen levels of 0-15 pO₂ mmHg; a        level equivalent to that present in hypoxic/necrotic sites in        human tumours). This hypoxia is produced by the inability of        oxygen to diffuse into the central areas of spheroid. The        glucose profile of the spheroid is similar to that seen for        oxygen.    -   FIG. 5B shows two different tumour spheroids (made of the breast        cancer cell line, MCF-7) following co-culture for 24 h with the        monocytic cell line, U937. The U937 cells (darkly stained cells        labelled with a monoclonal antibody to the pan-macrophage        marker, CD68) accumulate in the hypoxic rim of viable, but        hypoxic tumour cells around the central areas of necrosis (“N”).    -   FIG. 5C shows the infiltration into tumour spheroids of U937        cells preloaded with fluorescent dye. The top panel is a light        micrograph showing the opaque central area of necrosis (“N”)        which forms in these spheroids as a consequence of nutrient        (e.g. oxygen, glucose etc) deprivation. The bottom panel is the        same spheroid a fluorescent microscope to show the presence        within the spheroid of the fluorescent (i.e. light coloured        cells) U937 cells. The latter take up a similar position to that        seen in FIG. 5B, i.e. they congregate in a collar of hypoxic        tumour cells around the central areas of necrosis.

FIG. 6 illustrates cloning of HRE's in a receptor plasmid.

Table 1 represents specific examples of drug conjugates.

Materials and Method

A preferred way of working the invention will now be described withparticular reference to the use of mononuclear phagocytes to targettherapeutic genes to hypoxic sites in human tumours. However, it will beunderstood, that this illustration of the invention ie by way of usingmononuclear phagocytes to target therapeutic gene delivery is just oneway in which the invention may be worked. Moreover, this example of theinvention ie the targeting of therapeutic agents to human tumours is,yet again, just one example of how the invention may be worked.

EXAMPLE 1 Gene Transfer into Monocytic Cell Lines

Retroviral Transfer into the Monocytic Cell Line U937.

Techniques for retroviral vector mediated gene transfer into U937 cells(obtained from ATCC) were optimised with the marker gene β-galactosidaseusing retroviral particles packaged with two different envelopes.Retroviral constructs were packaged in a transient three plasmid system(Soneoka et al 1995 NAR 23 628-633); pHIT111 CMV lac Z genome with the4070A amphotropic envelope (Soneoka et al) and the MFG CMV nuclearlocalisation sequence lac Z genome pseudotyped with VSV-G (Cosset et alJ Virol 1995 69 7430-7436). Retroviral vectors were transduced into U937at multiplicity of infection of 1-3 in the presence of 8 μg/ml polybrenein maintenance media (RPMI/10% FCS, obtained from Sigma) for 2 h at 37°C. Cells were stained using standard X-gal histochemical techniques 48hlater (MacGregor et al 1991 p217-235, Methods in Molecular Biology, Vol7 Ed E J Murray). Levels of transduction were approximately 0.5 % withthe 4070A amphotropic envelope and approximately 1% pseudotyped withVSV-G. Transduction approaches may be increased using repeattransduction with high titre viral preparations concentrated usingstandard cross-flow filtration methods (following manufacturers protocolsupplied by Flowgen) and co-culture with producer cell lines(Morgenstern and Land 181-206, Methods in Molecular Biology, Vol 7 Ed EJ Murray). Cells transduced with pHIT111 were selected in 1 mg/ml G418(Gibco) resulting in the isolation of stable β-galactosidase positivecell lines as determined by X gal staining. FACS based selectionapproaches have been established using the fluoresceinatedβ-galactosidase substrate FDG (fluorescein di-b-D-galactoside) accordingto the manufacturers protocol (Molecular Probes).

Construction of Hypoxically Regulated Retroviral Genome.

To produce a hypoxically regulatable retroviral construct long aterminal repeat (LTR) plasmid was generated from the retroviral plasmidpLNSX (Miller and Rosman 1989 Biotechniques 7 980-990) by cutting at theNhel sites within the LTR, removing the majority of the interveningretroviral genome sequences and religating the backbone. This producesan LTR plasmid in which enhancer and enhancer/promoter swaps can beengineered. The retroviral enhancer was exchanged with the PGK hypoxiaresponse element (HRE) by performing an Nhe1/Xba1 swap. The resultantvector was then recut with Nhe1 and the Nhe 1 fragment of the retroviralgenome designated MOI was inserted. MOI was generated from the MFGvector (Bandara et al 1993 PNAS 90 10764-10768) by the generation of aminimal functional packaging signal using PCR with the followingprimers:

HindIIIR: GCATTAAAGCTTTTGCTCT

L523: GCCTCGAGCAAAAATTCAGACGGA

The fragment generated contains MMLV nucleotides +1-+523 and thus doesnot contain gag coding sequences that start at +621 (numbering accordingto Shinnick et al 1981 Nature 293 543-548). This fragment can beisolated as a HindIII/Xho1 fragment and used to replace thecorresponding region of MFG thus removing gag coding sequences. Thevector also includes the SD and SA sites spanning the intron of MFG andand FMDV internal ribosome entry site (IRES) sequence to allowcoincidental translation of two coding sequences from a singlebicistronic mRNA. A nuclear localising lac Z (including the nlssequences from SV40 large T antigen; Bonneret 1987 PNAS 84 6795-6799)was cloned immediately 3′ to this in the Stu1/Xho1 sites.

Electroporation of Monocytic Cell Lines

Electroporation conditions were optimised on the U937 and THP-1(obtained from ATCC) monocytic cell lines using β-galactosidase andluciferase reporter constructs based on the pGL3 vector series(Promega).

Optimal conditions using the Bio Rad Gene Pulser II were determined asfollows;

1 ×10⁷ cells were washed and resuspended in 500 μ1 serum free RPMI 1640(Sigma) at room temperature then transferred to a 0.4 cm electroporationcuvette. 30 μg DNA was added and the cuvette pulsed at 300V, 950 uFgiving time constants of between 20-25 milliseconds. Cells were restedat room temperature for 10 mins then seeded into 2 ml of RPMI/10% FCS(Sigma) in a 6-well plate and incubated at 37° C., 5% CO₂.

Transfection efficiencies were assayed by standard X-gal histochemistry(MacGregor et al 1991 p217-235, Methods in Molecular Biology, Vol 7 Ed EJ Murray) and Dual reporter luciferase assay (according to manufacturersprotocol obtained from Promega). Efficiencies were typically 2-5% forTHP-1 and 8-14% for U937. Stable transfectants were selected either byculture in 1 mg/ml G418 (Gibco) or using FDG FACS sorting (see above).

EXAMPLE 2 Gene Transfer to Primary Human Macrophages

Primary human macrophages were isolated from leukocyte enriched blood(obtained from the Blood Transfusion Service, Southmead Rd, Bristol) asfollows; peripheral blood mononuclear cells were obtained bycentrifugation through a Ficoll gradient (Pharmacia) according to themanufacturer's instructions. Macrophages were obtained from this cellpopulation by adherence on tissue culture plastic over 7 days in RPMI1640 (Dutch modified, Sigrrma) +2% heat inactivated human AB serum(Sigma) or 10% FCS. In some experiments, isolated macrophages weresubsequently cultured with the addition of 100 U/ml GM-CSF (obtainedfrom R and D systems).

Recombinant Adenovirus Mediated Gene Transfer to Primary HumanMacrophages.

Recombinant adenoviruses were created using the Quantum BiotechnologiesAdenoquest™ Kit (NBL Gene Sciences) according to the manufacturersinstructions. Briefly, the cDNA/reporter construct of interest (e.ghypoxia response element nls LacZ reporter cassette) was cloned into thepADBN transfer vector (FIG. 4) The vector was linearised near theInverted Terminal Repeat (ITR) to ensure efficient replication of therecombinant virus and was co-transfected along with the long arm of theadenoviral vector (QBI-viral DNA) into human 293 cells. Recombination in293 cells between the homologous regions of linearised transfer vectorand adenovirus genome vector produced complete adenoviral recombinants.The recombinants formed plaques in the 293 monolayer which were pickedas agarose plugs, purified by plaque assay and Arklone P centrifugationand characterised by PCR (Protocols for gene transfer in neuroscience:

Towards gene therapy of neurological disorders, Chapter 8, Ed.Lowenstein & Enquist (1996) Wiley & Sons). An example of such arecombinant adenovirus is QBI-HRE-LacZ shown in FIG. 4.

Recombinant adenovirus, QBI-AdenoLacZ, containing the CMV LacZ reporterconstruct (4×108 PFU/ml, Quantum Biotechnologies Adenoquest™ Kit, NBLGene Sciences) was used to infect primary macrophages seeded at adensity of 7×10⁵/ well in a 12 well plate. A multiplicity of infectionof 80 gave an infection efficiency of 10-20% after incubation for 24hours in the standard macrophage culture medium.

Gene Transfer to Cultured Primary Macrophages Using MannosylatedPolyLysine.

Differentiated macrophages express several cell-surfacelectins/receptors including the mannose-6-phosphate receptor (Shepherdet al. 1982 J. Reticuloendothel. Soc 32: 423-431). Primary macrophagescultured for 7-14 days in the presence of GM-CSF (see above) weretransfected using the pGL3 control plasmid (obtained from Promega withthe luciferase coding sequence replaced by that for β-galactosidase)using mannosylated poly-L lysine:DNA complexes according to publishedmethods (P. Erbacher et al, Human Gene Therapy 7: 721-729). This yielded20% transfectants as determined by X-gal histochemistry (MacGregor et al1991 p217-235, Methods in Molecular Biology, Vol 7 Ed E J Murray).

Construction of Plasmids Containing HREs

Sequences from a region approx. 300-375 bp upstrearn of thetranscription start of the human Enolase A gene were chosen containingthree HIF-1 consensus binding sites (Semenza et al 1996 J. Biol. Chem.271: 32529-32537. The following oligonucleotides were synthesised toassemble this sequence and add a BgIII site at the 5′ end and a BamHIsite at the 3′ end.

Lead strand (75-mer) GATCTGAGGGCCGGACGTGGGGCCCCAGAGCGACGCTGAGTGCGTGCGGGACTCGGAGTACGTGACGGAGCC CCG Complementary strand (75-mer)GATCCGGGGCTCCGTCACGTACTCCGAGTCCCGCACGCACTCAGCGTCGCTCTGGGGCCCCACGTCCGGCCC TCA

The oligonuclotides were inserted into the pGL3-pro vector (Promega) atthe BgIII site (see FIG. 7). Insertion of these oligonucleotides at thissite leaves a unique BamHI site downstream of the luciferase gene forinsertion of additional transcription units.For example, a transcriptionunit encoding HIF-1 alpha can be inserted at this site in order tofurther enhance the response to hypoxia.

Similarly, oligonucleotides were synthesised representing the HRE fromthe murine LDH gene (Firth et al 1995 J. Biol. Chem. 270: 21021-21027.The sequence chosen lies 15 bp upstream of the LDH TATAA box. Itcontains a HIF1 consensus binding site and a putative cyclicAMP-response element. This HRE, was also introduced at the BgIII site ofpGL3-pro.

Lead strand (56-mer) GATCTCTACACGTGGGTTCCCGCACGTCCGCTGGGCTCCCACTCTGACGTCAGCGG Complementary strand (56-mer)GATCCCGCTGACGTCAGAGTGGGAGCCCAGCGGACGTGCGGGAACC CACGTGTAGA

Cloning of the Human grp78 Promoter

The promoter was cloned by PCR amplification from human genomic DNA andincludes the complete promoter/enhancer sequence and 5′ UTR of the GRP78gene Chuck et al (1992). Nucleic Acids Research. 20: 6481-64; EMBL DataLibrary accession no. for the GRP78 promoter sequence: X59969). Theamplified fragment corresponds to bases 6-585 of this sequence. Theprimers used in the amplification reaction incorporated an Asel site atthe 5′ end and a XhoI site at the 3′ end. The grp78 promoter fragmentwas then cloned into these sites present in a Clontech pEGFP-N1 vectorallowing expression of beta-galactosidase/GFP fusion protein.

EXAMPLE 3 Infiltration of Multi-cellular Human Tumour Spheroids withHuman Macrophages (U937 Cells or Monocytes)

Tumour spheroids were established in culture using the MCF-7 cell line(ATCC) using the following procedure.

-   A. Establishment of Spheroid Cultures    -   1. Uniformly sized spheroids were grown in standard 96-well        tissue culture plates.    -   2. A 1.5% solution of agarose was prepared in media and        autoclaved (the media should not contain any supplements or        foetal calf serum as this causes the formation of bubbles and        the cells will plate down and not form spheroids).    -   3. 100 μl of the agarose was aliquoted into each well and        allowed to cool.        -   Plates were then warmed to 37° C. before use.    -   4. Monolayers of tumour cell lines were stripped in the        exponential growth phase, resuspended and counted using a        haemocytometer. The cells were then diluted to the appropriate        number of cells for spheroid initiation. For T47D and HT29 this        was 1000 cells per well and for MCF-7 it was 2000 cells per        well. Each well was filled with 200 μl of the cell suspension.        The final concentration of the cell suspension for T47D was 5000        cells per ml. (NB spheroids were grown in the media used        normally for each of the cell lines (eg T47D are grown in DMEM        supplemented with antibiotics and fungicides).    -   5. Following initiation, the spheroids were incubated at 37° C.        in a CO₂ incubator and left undisturbed for 5 days to allow        aggregation to occur.    -   6. Spheroids were fed fresh medium three times per week.-   B. Co-culture of Spheroids with Macrophages    -   1. Monocytic cell lines (eg U937 cells), peripheral blood        monocytes or monocyte-derived macrophages were introduced into        the spheroid culture once the spheroids have formed necrotic        centres. This stage depends upon the cell line used. For MCF-7        and T47D it was after 2-3 weeks of culture, when a dark area can        be seen in the centre of the spheroid.    -   2. This was done by removing 100 μl of media from the wells and        replacing it with a suspension of macrophages (50,000 per well).        For cell lines, the change in media was not a problem but for        PMBC the media should be serially changed for both the spheroid        and the macrophage until they are in the same media. These cells        infiltrated the spheroids in the first hour of co-culture and        continued to do so for up to 48 hrs. After this, the spheroids        were removed from the wells using a glass Pasteur pipette,        placed in a test tube and rinsed in PBS to remove any loose        macrophages or cell debris.    -   3. The spheroids were then allowed to settle to the bottom of        the tube, the PBS removed and the spheroids processed for        paraffin embedding (or frozen in OCT).

The results of an experiment showing infiltration of MCF-7 spheroidswith U937 cells is shown in FIG. 3. Each spheroid displays the typicalcentral area of necrosis (‘N’) surrounded by a collar of hypoxic tumourcells (approx 5-10 cells in thickness) and then several outer layers ofcells that are relatively normoxic. U937 cells, immuno-labelled (darkstaining) for the pan macrophage marker CD68, can be seen accumulatingin the hypoxic tumour cell layers around the central necrosis.

EXAMPLE 4 Demonstration of Infiltration into Spheroids of MonocyticCells Labelled with a Fluorescent Dye

U937 cells were incubated with 90 μl of4-(4-(didecylamino)styryl)-N-methyl pyridium iodide (2 mg/ml in absethanol) for 45 minutes then washed to remove the excess dye. Multicellspheroids (5-600 μm in diameter) were placed in a bacterial petri dish(agarose would fluoresce and they would adhere to tissue cultureplastic) and incubated with 75000 dye-loaded U937 cells per spheroid.The final volume was 15 mls. After 4 days of co-culture, spheroids werewashed to remove unattached macrophages and photographed using afluorescence microscope.

The result is shown in FIG. 6. Panel A in FIG. 6 shows a section througha spheroid photographed under white light. The central necrotic core (N)is visible as a dark area. Panel B, photographed under fluorescenceoptics, shows accumulation of macrophages labelled with fluorescent dye,in the hypoxic region around the necrotic core.

-   C. Paraffin embedding of spheroids    -   1. Spheroids were immersed in formalin for 2 hrs or overnight.    -   2. They were then either embedded in agar and processed or        placed in a piece of pre-folded tissue paper which was then        folded and placed in a tissue-processing cassette. Alternatively        a Cellsafe biopsy cassette was used (a mesh chamber in which the        spheroids are placed). The cassette was then closed and placed        inside the processing cassette. The mesh prevented the spheroids        escaping.    -   3. The spheroid preparations were processed through ascending        grades of alcohol to paraffin wax using a Citadel 2000        Histopathology Processing Unit.    -   4. Sections of the paraffin wax blocks were cut using a        microtome onto coated slides.-   D. Immunohistochemistry for CD68 (a pan macrophage marker) to    localise macrophages in spheroids    -   1. Spheroid sections were dewaxed in xylene and absolute        alcohol.    -   2. The endogenous peroxidase in the sections was then blocked        with 2% H202 in methanol for 10 mins.

3. Antigen retrieval: sections were exposed to proteinases type XXIV for15 mins at 37° C.

-   -   -   The sections were then incubated in the following: (with 3×5            min washes between each step)

    -   4. Normal serum for 30 mins at room temperature

    -   5. Primary anti-CD68 monoclonal antisera at a dilution of 1:100        for 1 hr at room temperature or overnight at 40° C.

    -   6. Secondary antibody (biotinylated horse anti-mouse IgG) for 30        mins at room temperature followed by an avidin-biotin-peroxidase        complex for 30 minutes at room temperature (ie using the Vector        ABC Elite kit)

    -   7. Visualized with the chrornagen, DAB or AEC for 20 10 mins.

    -   8. The nuclei were then counterstained with haematoxylin and        sections mounted with coverslips for viewing.        -   NB Spheroid sections needed to be washed thoroughly in            diluent between each step of the staining protocol to limit            background/non-specific staning.

EXAMPLE 5 Effect of Clamp-induced Hypoxia on Macrophage Infiltrationinto Tumour Xenografts

In order to determine the effect of hypoxia on macrophage infiltrationinto turnours in vivo, a xenograft model was established in which ahuman breast-tumour derived cell line is grown under the skin in nudemice. Clamping of the tumours induces increased hypoxia in the tumours.

-   A. Implantation and clamping of breast tumour xenografts in nude    mice    -   (as described in Griffiths L et al 1997 Cancer Res 57: 570-72)    -   1. Male nu/nu mice were injected s.c. with 5×107 MDA-231 cells        in the dorsal area in a volume of 0.1 ml. Twelve animals were        used per experimental group.    -   2. When the tumours reached 600 mm³ (ie after 6-7 weeks of        growth) they were clamped for 2 h to occlude the blood supply        and induce radiobiological hypoxia (<1% oxygen). Control tumours        were not clamped. (NB this procedure only partially occludes the        blood supply to the tumour, thereby allowing continued        macrophage infiltration into the tumour).    -   3. Tumours were excised immediately following clamp removal,        fixed and paraffin-embedded for routine histology and/or        immunohistochemistry for the pan murine macrophage marker,        F4/80.    -   B. Immunohistochemistry for the pan murine macrophage marker.        F4/80    -   1. Sections were dewaxed and rehydrated through graded alcohols        to distilled water.    -   2. Antigen retrieval: Serotec's reagent, STUF, was used to        unmask antigen (as described by the manufacturers).    -   3. Slides were then washed in PBS/0.1% Trition-X-100: 3        changes×5 mins each.    -   4. Endogenous peroxidase was blocked in sections in 2%        H₂O₂/methanol for 20 mins.    -   5. Sections were then washed as in Step 3.    -   6. Non specific protein binding sites in sections were blocked        using 5% normal rabbit serum in PBS for 30 mins.    -   7. Sections were then incubed in Serotec's F4/80 diluted to 1:20        in normal rabbit serum (5% NRS in PBS) for 90 mins at RT.    -   8. Sections were then washed as in Step 3.    -   9. The Vector biotinylated rabbit anti-rat (mouse absorbed) at        1:100 in PBS was then placed on the sections for 45 mins at RT.    -   10. Sections were then washed as in Step 3.    -   11. Sections were then incubated in Vector ABC reagent for 45        mins at RT.    -   12. Sections were then washed in Step 3.    -   13. The brown colour reaction was then developed with Vector DAB        kit for 2 mins.    -   14. The sections were then washed in water and nuclei was        counterstained in haematoxylin.    -   15. After dehydrating through alcohols, cleared in Xylene and        mounted with coverslips, the number of F4/80 positive        macrophages was then estimated for each tumour by taking the        average of the counts from 20, randomly selected fields of view        (×20 objective) obtained using a SeeScan image analysing device.

The results of this experiment are shown in FIG. 3. Clamping of thetumours led to increased infiltration of macrophages, demonstrating acorrelation between the degree of hypoxia and the number of infiltratingmacrophages.

Further Examples of Therapeutic Compositions For use in Working theInvention

Table 1 represents examples of drug conjugates for attaching to, orinternalising in, mononuclear phagocytes.

The drug conjugate of choice can be infused (repeatedly or as a singleinjection) into the general circulation so as to bind in vivo to thesurface of systemic mononuclear phagocytes and/or macrophages alreadyresident in diseased tissues (e.g. malignant tumours). In support of theaforementioned mode of drug delivery is the finding that when amonoclonal antibody, specific for the Mac-1 antigen on macrophages, isconjugated to indium (a radioactive element) and injected intravenouslyinto mice bearing a solid tumour, it was seen on scintographs toaccumulate predominantly in the cancer lesion (9). Alternatively thedrug conjugate can be exposed to monocytes ex vivo, following theirpurification from the blood of patients using such standard methods asFicoll-Hypaque gradients and elutriation as described previously in(10).

Homing of blood monocytes loaded up with drug conjugates into malignanttumours can be augmented by prior treatment with conventional systemictherapies which induce local inflammation/necrosis in the diseasedtissue (e.g. radiotherapy or chemotherapy in the case of cancerpatients). This stimulates the release of chemoattractant factors formonocytes/macrophages such as MCP-b 1 (11, 12) and would thus enhancethe delivery and hence the therapeutic efficacy of the drug conjugate atthe diseased site.

Mode ofproduction of selected drug conjugates

Drug 1. (with reference to Table 1) RSU1069-F(ab)₂ of a monoclonalantibody to CD87 (uPAR)

This conjugate uses a highly specific F(ab)₂ fragment a monoclonalantibody to CD87 (urokinase plasminogen activator receptor; uPAR) totarget naturally occurring uPAR on the surface of monocytes andmacrophages.

A monoclonal antibody to CD87 is made as described in (13) and thencleaved/purified to a specific F(ab)₂ monoclonal antibody fragment usingstandard proteolytic methods. Depending upon the part of uPAR used toraise the antibody (i.e. as the antigen), the epitope for the antibodygenerated may either be in the (i) ligand (i.e. uPA)-binding portion ofthe uPAR (in which case the drug conjugate will only bind to unoccupieduPAR on monocytes/macrophages), or (ii) the non ligand (i.e.uPA)-binding portion of the uPAR (in which case the drug conjugate willbind to both unoccupied and unoccupied UPAR on monocytes/macrophages).The most effective drug uptake is likely to be achieved using the latterform of conjugate.

The fragment of the CD87 monoclonal antibody is conjugated to thebioreducative prodrug, RSU1069, by the latter being reacted with anagent such as arylazide to add a N-hydroxysuccinimide group. This isthen cross-linked at neutral pH to the CD87 antibody fragment via aminegroups to form a conjugate. This method is well established forconjugating drugs to proteins and is described fully in (14).

Drug 2. (with reference to Table 1) i.e. RSU1069-PAI-2

This conjugate uses the affinity of plasminogen activator inhibitor 2(PAI-2) for urokinase plasminogen activator receptor (uPAR)—urokinaseplasminogen activator complexes to target the bioreductive prodrug tothe surface of monocytes and macrophages. PAI-2 triggers theinternalization of uPAR-uPA complexes, so the internalization by thesecells of the bioreductive prodrug attached to PAI-2 is assured.

Naturally occurring PAI-2 is obtained from the culture supernatant ofhuman blood monocytes stimulated maximally with interleukin 1 or 2 asdescribed in (15). This is then purified to homogeneity in the usualmanner by elution from an anti-PAI-2 immunoaffinity column.Alternatively, PAI-2 can be produced in a recombinant expression systemand purified according to the method of (16). The PAI-2 preparation isthen conjugated to RSU1069 using the method outlined as hereinbeforedescribed for drug 1(14).

Drug 6. (with reference to Table 1) i.e. Interleukin-2 (IL-2) genelinked to hypoxia responsive promoter.

This gene is transferred to monocytes/macrophages using areplication-defective adenoviral vector. Efficient transfer of genesinto human macrophages has been achieved with this method withexpression of the gene in 40-80% of the cells exposed to the vector andlasting up to 3 weeks after gene transfer (10).

Defective retroviral vectors, direct DNA internalization or suchnon-gene viral gene transfer systems can also be used such as cationiclipids, liposomes, lectins or polymers. Genes other than IL-2 whichcould be of therapeutic benefit include such other immunomodlators asTNFá, or interferon gamma, prodrug activating enzymes, enzymeinhibitors, tumour antigens (to provoke the hosts immune reactivity tothe tumour) and anti-oncogenes (antibodies or antisense RNA). It will beunderstood by those skilled in the art that the DNA construct used may,embody a number of these genes rather than just one and is not intendedto limit the scope of the application.

The hypoxically inducible expression control sequence (promoter) for theEpo or PGK genes (or multiple copies thereof) is/are coupled to one ormore of the gene sequences of choice (e.g. IL-2 gene sequence) asdescribed by one of us previously in (17).

Ex vivo gene transfer:

The method outlined in (10) involves incubating (under sterileconditions) freshly isolated blood monocytes or monocyte-derivedmacrophages (monocytes incubated overnight in teflon bags or on plasticcultured wells) in the presence of 100 plaque-forming units per cell ofthe purified replication-defective vector, Ad.RSVâ, harbouring thehypoxia-responsive promoter-II-2 gene construct in RPM1 incubationmedium (1 ml/10⁶ cells). Gene transfer may be enhanced by simultaneoustreatment of the cells with 10 U/ml of human interferon gamma. Cells arethen washed to remove free viral particles and interferon gamma andreincubated at 37° C. in fresh RPMI medium in teflon bags. The sameadenoviral vector but harbouring the E.coli â-galactosidase gene(Ad.RSV.âgal) instead of the hypoxia-responsive promoter-IL-2 geneconstruct is used as a reporter gene (i.e. to check the efficiency ofthis gene transfer method to monocytes/macrophages ex vivo). Thepresence of the â-gal enzyme in cells after infection with Ad.RSV.âgalis then assessed using histochemical methods as described in (17). Thetransfected cells (10⁸ to 10⁹ cells) are then injected sterile back intothe bloodstream or directly into the appropriate diseased tissue (e.g.malignant tumour) of the donor as in (10).

In vivo gene transfer:

This is performed according to the in vivo method of adenoviral genedelivery described in (18). This involves injecting the vector (i.e.Ad.RSVâ) bearing the hypoxia-responsive promoter-IL-2 gene constructinto the bloodstream (primarily to label monocytes) and/or into thediseased tissue (at 10⁹ to 10⁷ plaque-forming units) to label tissuemacrophages.

The invention is exemplified by the drug conjugate candidates inTable 1. It will be understood by those skilled in the art that suchconjugates represent selected examples and are not intended to limit thescope of the invention, furthermore it will be understood that indeedany one example of a candidate Part I drug conjugate may, whereappropriate, be used in conjunction with any one example of a Part IIcandidate and/or any example of a Part III candidate. Additionally itwill be understood that any example of a Part III candidate may be usedin conjunction with any example of a Part II and/or Part I candidatedrug conjugate.

The invention hereinbefore described therefore represents a most elegantand effective means and method of delivering a therapeutic and/orcytotoxic agent to a hypoxic or ischaemic or stress site by use ofmonocytes and/or macrophones and their natural ability to congregate ata hypoxic site.

TABLE 1 SPECIFIC EXAMPLES OF DRUG CONJUGATES Part 1 Part II Part IIIDrug 1 RSU1069 F(ab)2 to — or CD87 (uPAR) Tirapazamine Drug 2 RSU1069PAI-2 — or (binds to Tirapazamine receptor bound urokinase plasminogenactivator (uPAR) & ensures internali- zation of drug) Drug 3 RSU1069PAI-2 NADPH:P450 or (binds to reductase Tirapazamine receptor (reductasefor bound activation of uPAR & ensures RSU1069 or internalizationTirapazamine of drug) under hypoxia) Drug 4 RSU1069 F(ab)2 to NADP:P450or CSF-1 reductase Tirapazamine receptor (binds to human CSF receptor onsurface of monocyte/ macrophages) Drug 5 Cytosine F(ab)2 to CD63 —deaminase (prodrug activating enzyme) Drug 6 Gene for Inter- Hypoxia.leukin 2 or ischaemia (immuno- or stress stimulatory -resp. promotercytokine) in sequence Ad.RSV Drug 7 DNA sequence — Hypoxia. for solubleor ischaemia domain of VEGF or stress receptor (eg. flk-1) -resp.promoter in Ad. RSV sequence Drug 8 A therapeutic gene pGL3-pro Hypoxiafor example as plasmid and or ischaemia described in mannosylated orstress PCT/GB95/00322 poly-L lysine -resp. promoter W09521927 sequenceDrug 9 A therapeutic gene Adenoviral vector Hypoxia for example as forexample or ischaemia described in QBI-HRE-LacZ or stress PCT/GB95/00322-resp. promoter WO9521927 sequence

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1. A mononuclear phagocyte modified to comprise at least one regulatable element operably linked to at least one nucleotide sequence of interest (NOI), wherein said regulatable element is capable of regulating expression of said NOI in a tumour site and is selected from a hypoxia regulatable element, an ischemic regulatable element and a stress regulatable element.
 2. The mononuclear phagocyte according to claim 1 wherein the mononuclear phagocyte comprises a binding agent capable of binding to a cell surface element of the mononuclear phagocyte.
 3. The mononuclear phagocyte according to claim 2 wherein the binding agent comprises a mannosylated poly-L-lysine ligand.
 4. The mononuclear phagocyte according to claim 2 wherein the binding agent comprises a viral vector for internalising the regulatable element into the mononuclear phagocyte.
 5. The mononuclear phagocyte according to claim 1 wherein the NOI is incorporated into the genome of the mononuclear phagocyte.
 6. The mononuclear phagocyte according to claim 4 wherein the viral vector is a lentiviral vector.
 7. The mononuclear phagocyte according to claim 1 wherein the mononuclear phagocyte further comprises an NOI encoding HIF1-alpha or a tetracycline repressor protein.
 8. The mononuclear phagocyte according to claim 1 wherein the at least one NOI encodes a pro-drug activation enzyme.
 9. A construct comprising at least one regulatable element operably linked to at least one nucleotide sequence of interest (NOI), wherein said regulatable element is selected from a hypoxia regulatable element, an ischemic regulatable element and a stress regulatable element, and wherein the construct is coupled to a binding agent tat is capable of selective binding to a cell surface element of a mononuclear phagocyte.
 10. The construct according to claim 9 wherein the regulatable element is an HRE element.
 11. The construct according to claim 9 or claim 10 wherein the binding agent comprises a ligand adapted to bind to the cell surface element.
 12. The construct according to claim 9 or claim 10 wherein the binding agent comprises a viral vector for internalising the regulatable element into a mononuclear phagocyte.
 13. The construct according to claim 12 wherein the viral vector is selected from the group consisting of an adenoviral vector and a lentiviral vector.
 14. A method for internalising a regulatable element into a mononuclear phagocyte wherein the regulatable element is selected from a hypoxia regulatable element, an ischemic regulatable element and a stress regulatable element and the method comprises: providing a mononuclear phagocyte ex vivo; and exposing the mononuclear phagocyte to a construct as defined in any one of claims 9 or 10 under conditions sufficient to internalise the construct into the mononuclear phagocyte.
 15. A composition comprising a mononuclear phagocyte according to claim 1 optionally admixed with a pharmaceutically acceptable diluent, excipient or carrier.
 16. A composition comprising a construct according to claim 9 optionally admixed with a pharmaceutically acceptable diluent, excipient or carrier.
 17. A mononuclear phagocyte comprising an NOI encoding a p450 enzyme wherein the NOI has been internalised into the mononuclear phagocyte by an adenoviral vector; and wherein the NOI encoding the p450 enzyme is operably linked to a hypoxia response element (HRE); such that the p450 enzyme is expressed under suitable hypoxic conditions.
 18. The mononuclear phagocyte according to claim 8 wherein the pro-drug activation enzyme is a p450 enzyme.
 19. The mononuclear phagocyte according to claim 18 wherein the p450 enzyme, is a CYP2B6 p450 enzyme.
 20. The construct according to claim 11 wherein the ligand is a mannosylated poly-L-lysine. 